Abstract T cell-based therapies have emerged recently as important therapies for cancer. Resistance to the activity of these cells, relapse, toxicities and deaths are still important hurdles to their success and strongly call for mechanisms to potentiate efficacy, while at the same time, to better control the safety of cell therapies. The goal of this project is to create a new generation of synthetic, tumor-specific T cells (based on CAR and TCRm technology) that will selectively home to cancer cells in vivo and then synthesize and release anti-neoplastic drugs at the cancer cell surface or into the tumor microenvironment. The selective and local elaboration of potent anti-neoplastic drugs at the tumor site, or on the cancer cells, should simultaneously: 1) reduce toxicity relative to conventional cancer drug therapy; 2) overcome immune mediated resistance to conventional CAR T cell therapy from regulatory cells and cytokines in the tumor microenvironment (because the cytotoxic drugs made will not be affected;) 3) overcome antigen loss variant mechanisms of resistance (because the drugs will kill cancer cells without antigen on the cell surface;) 4) reduce toxicity relative to conventional CAR T cells because the prodrug infusions can be pharmacologically regulated, scheduled, or stopped. In addition, the enzyme expression can be made conditional. The components of the proposed strategy are: 1) prodrug/drug pair in which the prodrug is not toxic to normal cells or tissues and the resulting drug potently kills cancer cells. Several types of prodrug systems will be developed for various functions and properties (Aim 1). 2) A CAR T cell directed to a tumor specific antigen via a lineage specific scFv (such as CD19 or MUC16) or a more specific TCRm-based scFv (such as to WT1 or PRAME,) 3) An enzyme genetically engineered into the CAR T cell capable of converting the prodrug into the active drug locally. We term these cells ?Synthetic Enzyme Armed KillER? cells or SEAKER cells. These cells will be designed and tested in vitro (Aim 2) and in animal models (Aim 3.) This work will be carried out through a multi-PI, multidisciplinary collaboration between the labs of David A. Scheinberg (PI), Derek S. Tan (Co-PI), and Renier Brentjens (Co-Investigator), comprising extensive expertise in synthetic and medicinal chemistry, biochemistry, pharmacology, cell biology, and cancer immunology, that builds on prior work from the leaders over 15 years.